CD8+ T cells recognize class I molecules of the major histocompatibility complex (MHC) bearing peptides of 8 to 10 residues derived from cytosolic proteins. These cells are a bulwark of host defenses to infectious agents and tumors, and if we are to improve existing vaccines and develop new vaccines and treatments for infectious and neoplastic diseases, it is critical to understand antigen processing, the mechanism by which antigenic peptides are generated by cells and delivered to class I molecules. Although there has been great progress in understanding antigen processing in the past five years, there remains much to be learned. The strategy of this project is to delineate what we do not know. We are expressing the known constituents of the antigen processing machinery in mosquito cells and determining whether this is sufficient to reconstitute antigen processing to a level seen in mammalian cells. Insects do not have a MHC and do not, therefore, have any of the specialized machinery associated with antigen processing. If these cells can process antigens under these conditions, this would indicate that we have identified the major components of antigen processing; if they can not, it would mean that further discoveries are required. In the past year, we found that class I molecules expressed in cells can bind to antigenic peptides targeted to the endoplasmic reticulum by a signal sequence, demonstrating that no special accessory proteins are essential for this process. On the other hand, this assembly is much less efficient than in mammalian cells, suggesting that there are specialized proteins that facilitate peptide loading onto class I molecules. Taken together these findings indicate that insect cells are capable of assembling functional class I molecules in the ER, but that this process is rather inefficient. This system should be useful for identifying and characterizing novel proteins involved in antigen processing.